The purpose of the proposed study is to define new processes that regulate phototransduction and adaptation in visual photoreceptors. The long-term goal is to determine the molecular mechanisms that regulate synthesis of a second messenger of phototransduction, cyclic GMP (cGMP), by retinal membrane guanylyl cyclases (retGC). The proposal is based on the finding that photoreceptor-specific calcium sensor proteins, GCAPs, that regulate retGC in response to the change in intracellular free calcium concentrations are essential for normal photoresponses and survival of photoreceptor cells. Recent evidence supports our hypothesis that magnesium binding in two EF-hand domains controls GCAP docking with the cyclase in the light. Therefore, the first aim of the proposal is to identify, by using directed mutagenesis, the docking sites in retGC and GCAPs and to test their function in vitro and in vivo. There are two ubiquitous GCAPs in vertebrates, GCAP1 and GCAP2. Recent findings using GCAP1 and GCAP2 gene knockout mouse models have lead to a hypothesis that activation of cGMP synthesis in response to light is a two-step process, in which GCAP1 and GCAP2 stimulate cGMP production sequentially, according to their different calcium sensitivity. There are also two isozymes of retGC, retGC1 and retGC2, required for rod recovery. Therefore, the second specific aim is to establish how the two different isozymes of retGC are regulated by GCAP1 and GCAP2 in mouse rods. We plan to establish parameters and biochemical regulation of individual retGCs by individual GCAP1 or GCAP2 and determine their contribution to the rod physiology and to the progression of photoreceptor cell death caused by mutations in GCAP1. Cone cells respond to light and recover faster than rods and adapt to light very rapidly, thus leading to a hypothesis that cGMP synthesis is regulated faster in cones than in rods. Therefore, the third specific aim of this proposal is to determine the biochemical properties of retGC in cones and the role of individual GCAPs in regulation of cone retGC activity. The proposed experiments are relevant to the understanding of the mechanisms that control photoreceptor activity and cause inherited retinal diseases in human patients. PUBLIC HEALTH RELEVANCE: The proposed study has relevance to public health, because its completion will provide better understanding of the processes important for both normal vision and visual disorders. The main goal for this research is to understand how retinal guanylyl cyclases and calcium-binding proteins control electrical responses of the retina and why the mutations that affect these proteins lead to congenital blindness. The specific purpose of this study is to elucidate molecular mechanisms of their interactions and their role in retinal physiology and retinal degeneration.